African swine fever virus (ASFV) is a highly pathogenic large DNA virus that causes African swine fever (ASF) in domestic pigs and wild boars. The p17 protein, encoded by the D117L gene, is a major transmembrane protein of the capsid and the inner lipid envelope. The aim of this study was to investigate the effects of p17 on cell proliferation and the underlying mechanisms of action. The effects of p17 on cell proliferation, cell cycle, apoptosis, oxidative stress, and endoplasmic reticulum (ER) stress have been examined in 293T, PK15, and PAM cells, respectively. The results showed that p17 reduced cell proliferation by causing cell cycle arrest at G2/M phase. Further, p17-induced oxidative stress and increased the level of intracellular reactive oxygen species (ROS). Decreasing the level of ROS partially reversed the cell cycle arrest and prevented the decrease of cell proliferation induced by p17 protein. In addition, p17-induced ER stress, and alleviating ER stress decreased the production of ROS and prevented the decrease of cell proliferation induced by p17. Taken together, this study suggests that p17 can inhibit cell proliferation through ER stress and ROS-mediated cell cycle arrest, which might implicate the involvement of p17 in ASF pathogenesis.